Activation and dysfunction of the endothelium is a critical factor in many vascular disorders including atherosclerosis, tumor growth, and inflammation. It is well-established that the forkhead transcription factor, FOXO1 functions critically to transduce external stimuli in the endothelium into changes in gene expression and cellular phenotype. FOXO1 responds to a diverse set of cellular cues to restrict the proliferative capacity of cells, repress cell survival, and activate apoptosis. Current data points to important functional roles for FOXO1 signaling in vascular functions and phenotypes in endothelial cells and suggest modulation of FOXO1 activity may have implications in cardiovascular disease. The structural details to fully explain the molecular basis of FOXO1 function and importantly physiochemical mechanisms that regulate FOXO1 interactions at an atomic level remain poorly defined. We hypothesize that the functions of the FOXO1 DNA-binding domain (DBD) and nuclear localization sequence region (NLS), FOXO1-DBD/NLS, are coordinated by the combinatorial action of domain protein structure, posttranslational modifications, and interactions with protein partners. These factors produce a 'physiochemical- code' that specifies FOXO1-dependent gene expression in response to stimuli in endothelial cells. Elucidating the molecular details of FOXO1 structure/function is critical to fully understanding its targeted transcription specificity and addressing critical gaps in our knowledge of how varied posttranslational modifications in the domain contribute to complex levels of regulation. The overall goal of this proposal is to leverage protein structure information from biophysical, NMR spectroscopy and X-ray crystallography studies to determine relationships between structure and function in the FOXO1-DBD/NLS, reveal those molecular mechanisms that define the 'physiochemical-code' of FOXO1 activity, and use these data to develop pharmacological/therapeutic modulators of FOXO1 activity. We will determine the structure of FOXO1-DBD/NLS free and in complex with its DNA recognition sequence using NMR to identify the structure-function importance of as yet structurally undetermined regions in FOXO1. We will determine the crystal structure of a novel ternary FOXO-Smad3-DNA complex to gain insight into FOXO-TF interactions in a transcriptional complex integrating key signaling pathways (Aim1). We propose to examine the FOXO1-DBD/DNA interface using in vitro binding experiments and NMR methods to understand the structure/function effects of varied posttranslational modifications, including effects on DNA-binding, domain flexibility, and protein interaction and assess the effects of posttranslational modifications on FOXO1 interactions and functions in endothelial cells (Aim 2). Finally, we will use this structural information to both rationally design antagonizing, cell-permeable peptide inhibitors of FOXO1-transcriptional activity and to computationally screen for small molecule inhibitors of FOXO1- DNA binding (Aim 3). These studies are significant in that they will yield novel targets for cardiovascular disease.